The cytokine known as tumor necrosis factor.alpha. (TNF.alpha.; also termed cachectin) is a protein secreted primarily by monocytes and macrophages as a soluble homotrimer of 17 kD protein subunits in response to endotoxin or other stimuli (Smith, R. A. et al., J. Biol. Chem. 1987, 262, 6951-6954). A membrane-bound 26 kD precursor form of TNF.alpha. has also been described (Kriegler, M. et al., Cell 1988, 53, 45-53). TNF.alpha. was originally discovered in the serum of animals injected sequentially with a bacterial vaccine (bacillus Calmette-Guerin, BCG) and endotoxin (Carswell, E. A. et al., Proc. Natl. Acad. Sci. U.S.A 1975, 72, 3666).
The expression of the gene encoding TNF.alpha. is not limited to cells of the monocyte/macrophage family. Several human non-monocytic tumor cell lines were shown to produce TNF.alpha.. TNF.alpha. is also produced by CD4.sup.+ and CD8.sup.+ peripheral blood T lymphocytes, and by various cultured T and B cell lines.
TNF.alpha. plays an integral role in destroying tumors, mediating responses to tissue injury, and protecting hosts from infections by various microorganisms (Goeddel et al., Cold Spring Harbor Symp. Quant. Biol. 1986, 51, 597-609; Beutler et al., Ann. Rev. Immunol. 1989, 7, 625-655; Malik et al. in Tumor Necrosis Factor: Structure, Function and Mechanism of Action, Aggarwal and Vilcek, Eds. (Marcel Dekker, Inc., 1992); Fiers, FEBS Letters 1991, 285, 199-212; and Buetler etal., Buetler, B., Ed. in Tumor Necrosis Factors: the Molecules and Their Emerging Role in Medicine (Raven Press, New York, N.Y., 1992)). However, its activity appears to be excessive in some disease states and inflammatory reactions such as rheumatoid arthritis, cachexia, and septic shock (Pujol-Borrell et al., Nature 1987 326, 304-306; Oliff, Cell 1988 54, 141-142; Tracey et al., Nature 1987, 330, 662-664). The excess TNF.alpha. results in an exaggerated immune response exemplified by overstimulation of interleukin-6 and granulocyte/macrophage-colony stimulating factor (GM-CSF) secretion, enhanced cytotoxicity of polymorphonuclear neutrophils, and prolonged expression of cellular adhesion molecules, all of which can have detrimental effects. The benefits of inhibiting TNF.alpha. activity during inflammatory reactions in animal models have been demonstrated using neutralizing monoclonal antibodies to TNF.alpha. (Tracey et al., Nature 1987, 330, 662-664; Silva et al., J. Infect. Sis. 1990, 162, 421-427; and Williams et al., Proc. Natl. Acad. Sci. 1992, 89, 9784-9788).
The mechanism of action of TNF.alpha. is derived from accumulating evidence which indicates that TNF.alpha. is a regulatory cytokine with pleiotropic biological activities. These activities include: inhibition of lipoprotein lipass synthesis ("cachectin"), activation of polymorphonuclear leukocytes, inhibition of cell growth or stimulation of cell growth, cytotoxic action on certain transformed cell types, antiviral activity, stimulation of bone resorption, stimulation of collagenase and prostaglandin E2 production, and immunoregulatory actions, including activation of T cells, B cells, monocytes, thymocytes, and stimulation of the cell-surface expression of major histocompatibility complex class I and class II molecules.
TNF.alpha. is noted for its pro-inflammatory actions which result in tissue injury, such as induction of procoagulant activity on vascular endothelial cells (Pober, J. S. et al., J. Immunol. 1986, 336, 1680), increased adherence of neutrophils and lymphocytes (Pober, J. S. et al., J. Immunol. 1987, 138, 3319), and stimulation of the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi, G. et al., J. Exp. Med. 1987, 166, 1390).
Recent evidence implicates TNF.alpha. in the pathogenesis of many infections (Cerami, A. et al., Immunol. Today 1988, 9, 28), immune disorders, neoplastic pathology, e.g., in cachexia accompanying some malignancies (Oliff, A. et al., Cell 1987, 50, 555), and in autoimmune pathologies and graft-versus host pathology (Piguet, P.-F. et al., J. Exp. Med. 1987, 166, 1280). The association of TNF.alpha. with cancer and infectious pathologies is often related to the host's catabolic state. A major problem in cancer patients is weight loss, usually associated with anorexia. The extensive wasting which results is known as "cachexia" (Kern, K. A. al., J. Parent. Enter. Nutr. 1988, 12, 286-298). Cachexia includes progressive weight loss, anorexia, and persistent erosion of body mass in response to a malignant growth. The fundamental physiological derangement may be related to a decline in food intake relative to energy expenditure. The cachectic state is thus associated with significant morbidity and is responsible for the majority of cancer mortality. A number of studies have suggested that TNF.alpha. is an important mediator of the cachexia in cancer, infectious pathology, and in other catabolic states.
TNF.alpha. is thought to play a central role in the pathophysiological consequences of Gram-negative sepsis and endotoxic shock (Michie, H. R. et al., Br. J. Surg. 1989, 76, 670-671; Debets, J. M. H. et al., Second Vienna Shock Forum, 1989, p. 463-466; Simpson, S. Q. et al., Crit. Care Clin. 1989, 5, 27-47), including fever, malaise, anorexia, and cachexia. Endotoxin is a potent monocyte/macrophage activator which stimulates production and secretion of TNF.alpha. (Kornbluth, S. K. et al., J. Immunol. 1986, 137, 2585-2591) and other cytokines. Because TNF.alpha. could mimic many biological effects of endotoxin, it was concluded to be a central mediator responsible for the clinical manifestations of endotoxin-related illness. TNF.alpha. and other monocyte-derived cytokines mediate the metabolic and neurohormonal responses to endotoxin (Michie, H. R. et al., N. Eng. J. Med. 1988, 318, 1481-1486). Endotoxin administration to human volunteers produces acute illness with flu-like symptoms including fever, tachycardia, increased metabolic rate and stress hormone release (Revhaug, A. et al., Arch. Surg. 1988, 123,162-170). Elevated levels of circulating TNF.alpha. have also been found in patients suffering from Gram-negative sepsis (Waage, A. et al., Lancet 1987, 1, 355-357). Treatment of cancer patients with TNF.alpha. (because of its tumoricidal action) revealed that doses greater than 545 .mu.g/m.sup.2 /24 hours caused alterations similar to those induced by injection of endotoxin (4 ng/kg) into healthy humans (Michie, H. R. et al., Surgery 1988, 104, 280-286), supporting TNF.alpha.'s role as the principal host mediator of septic and endotoxemic responses. Chronic intravenous TNF.alpha. infusion into humans or rats was associated with anorexia, fluid retention, acute phase responses, and negative nitrogen balance (i.e., classic catabolic effects), leading to the conclusion that TNF.alpha. may be responsible for many of the changes noted during critical illness (Michie, H. R. et al., Ann. Surg. 1989, 209, 19-24).
The numerous biological effects of TNF.alpha. and the closely related cytokine, TNF.beta. (lymphotoxin), are mediated by two transmembrane receptors, both of which have been cloned. The p55 receptor (also termed TNF-R55, TNF-RI, or TNFR.beta.) is a 55 kd glycoprotein shown to transduce signals resulting in cytotoxic, anti-viral, and proliferative activities of TNF.alpha..
The p75 receptor (also termed TNF-R75, TNF-RII, or TNFR.alpha.) is a 75 kd glycoprotein that has also been shown to transduce cytotoxic and proliferative signals as well as signals resulting in the secretion of GM-CSF. The extracellular domains of the two receptors are 28% identical in primary structure and have in common a set of four subdomains defined by numerous conserved cysteine residues. The p75 receptor differs, however, by having a region adjacent to the transmembrane domain that is rich in proline residues and contains sites for 0-linked glycosylation. Interestingly, the cytoplasmic domains of the two receptors share no apparent homology which is consistent with observations that they can transduce different signals to the interior of the cell.
TNF.alpha. inhibitors have been detected in normal human urine and in serum of patients with cancer or endotoxemia. These have since been shown to be the receptor extra-cellular domains derived by proteolytic cleavage of the transmembrane forms. Many of the same stimuli that result in TNF.alpha. release also result in the release of the soluble receptors, suggesting that these soluble TNF.alpha. inhibitors may serve as part of a negative feedback mechanism to control TNF.alpha. activity (Porteu, F. and C. Nathan (1990) J. Exp. Med. 172: 599-607; and, Adreke, D. et al., (1992) J. Exp. Med. 175: 323-329).
There is a need for compounds which effectively inhibit TNF.alpha. activity. There is a need to provide compounds that bind to TNF.alpha. with high affinity and can prevent TNF.alpha. from binding to its receptors. There is a need for compounds which can neutralize TNF.alpha. activity in vivo.